In the present study, we examined patients with esophageal cancer for an increase in the expression levels of 14 autoantibodies and further evaluated the effects of gene expression, mutation, and amplification on the expression of these autoantibodies using TCGA data. All antibodies, except VEGF, demonstrated a certain positive expression rate compared with control. In addition, there was little or no mutation or amplification for any gene. We believe that an increase in gene expression might be responsible for the increased expression of these antibodies. Among the 14 autoantibodies, LGALS1, HCA25a, HCC-22-5, and HSP70 demonstrated a relatively high expression level compared to that of control, and hence they were extracted for further investigation. The expression levels of CEA and SCC-Ag, conventional tumor markers, were evaluated and compared with control. An increased expression level of the four antibodies was not inferior to the combination of CEA and SCC. Furthermore, the combination of the panel and the tumor marker significantly improved the positive rate compared with their own positive rates.
In our previous studies, we observed that serum p53 antibodies appeared at a higher rate in patients with ESCC [19, 28]. TCGA data confirmed that 156 (68.7%) of 227 patients with ESCC have p53 gene mutations. According to previous reports, the positive rate of p53-Ab is considered to be approximately 20% in patients with early-stage esophageal cancer and ≥30% in patients with advanced cancer [19, 29]. TCGA data demonstrated the presence of p53 gene mutation irrespective of early or advanced cancer, and it appears that gene mutation is significantly involved in the appearance of p53 antibodies. This is another reason that p53 antibodies are detected in patients with early-stage cancer.
It is known that when gene mutation occurs in p53, there is an increase in the expression of abnormal genes and abnormal proteins. When an antibody appears, it is considered to be the result of a series of flaws from a gene mutation accompanied by the appearance of an abnormal gene or abnormal protein. In contrast, NY-ESO-1 antibodies have been reported in several types of cancers . Previous studies have reported positive expression rates of antibodies of 32.0%, 12.3%, 12.1%, 10.5%, 10.3%, 8.4%, and 7.1% for ESCC, lung cancer, liver cancer, prostate cancer, colon cancer, and breast cancer, respectively. However, according to TCGA data, the frequencies of gene mutation of each cancer type were 0%, 0.24%, 0.27%, 0.9%, 0.09%, 0.32%, and 0.09% and the gene amplification rates were 0%, 1.5%, 1.9%, 2.7%, 0.3%, 0.65%, and 0.8%, respectively. Even when both mutation rates are combined, the frequency ranges from 0% to several percentage. Gene expression did not increase in esophageal cancer and breast cancer, but increased expression was observed in other cancer types (P = 0.06 for esophageal cancer, with a tendency to increase). Sato et al. reported that gene expression levels were increased in ESCC and HCC . However, because the frequency of gene mutation and gene amplification is not high, a mechanism not associated with p53 may be involved in these gene expression increases. In malignant mesothelioma, the expression of NY-ESO-1 is increased through histone deacetylase inhibitor . Studies conducted using ovarian cancer cell lines have reported that the expression level of NY-ESO-1 was increased using DNA methylation inhibitors , indicating that epigenetic mechanisms such as acetylation and methylation may be involved in NY-ESO-1 expression. We found almost no gene mutation or amplification for the 14 antigens and antibodies that we selected in our study. Except for two antigens for which data could not be confirmed, and except for KM-HN-1, there was a significant increase in gene expression in the cancer tissue compared to that in the normal tissue. It is possible that mechanisms other than gene mutation and amplification were involved in increasing the expression and appearance of antigens.
We had previously reported that using antibody panels in gastric cancer can provide results that surpass the results provided by existing tumor markers . In the present study, we extracted four antibodies that are considered to have a relatively high positive rate among the 14 antibodies and evaluated them as a panel. We found that the positive rate intensely improved to 32.9%. Moreover, it was confirmed that the combination of CEA and SCC, conventional tumor markers, significantly increased the sensitivity compared with the use of the panel alone or the combination of tumor markers. Antibodies, which are considered to have an insufficient positive rate by themselves, may improve their usefulness by being added to a panel or when used in combination with conventional tumor markers. The specificity of any antibody was 100% at the cut-off value used in the present study in the current cohort, and their usefulness is regarded as appropriate considering that the specificity of existing tumor markers is not sufficiently high . In recent years, microsatellite instability, tumor mutational burden, and other genetic mutations have received attention as biomarkers for their effects of immune checkpoint inhibitors [35-37]. This is because they are believed to be involved in increasing the appearance of targeted neoantigens and cancer antigens. There is a possibility that the increase in antibodies we examined in the present study could correlate with an effect of immune checkpoint inhibitors; however, this assumption needs to be clarified in future clinical trials.
A limitation of the present study was that it was conducted at a single facility. However, the ongoing clinical trials in esophageal cancer and hepatocellular carcinoma, including studies on the antibodies we have examined in the present study, are being conducted in collaboration with other institutions. We anticipate that the usefulness of these antibodies will become clearer in the near future. Since data such as gene expression, mutation, and amplification were analyzed using TCGA public data, it is not the case used for autoantibody analysis in this study, and a detailed evaluation of differences among races and others was not possible.